Environmental Biology of 48: 127–155, 1997.  1997 Kluwer Academic Publishers. Printed in the Netherlands.

Phylogeny of the : cytogenetic and molecular approaches

Vadim J. Birstein1, Robert Hanner2 & Rob DeSalle3 1 The Society, 331 West 57th Street, Suite 159, New York, NY 10019, U.S.A. 2 Department of Biology, University of Oregon, Eugene, OR 97405–1210, U.S.A. 3 Department of Entomology, American Museum of Natural History, New York, NY 10024, U.S.A.

Received 20.3.1995 Accepted 21.3.1996

Key words: sturgeon, , , , , , Polyodon, Pse- phurus, karyotype, chromosome, macrochromosome, microchromosome, genome, DNA content, 18S rRNA gene, cytochrome b, 12S mtrRNA gene, 16s mtrRNA gene, rate of molecular , phylogeny, evolution

Synopsis

The review of the data on karyology and DNA content in Acipenseriformes shows that both extant families, the Polyodontidae and Acipenseridae, originated from a tetraploid ancestor which probably had a karyotype consisting of 120 macro- and microchromosomes and DNA content of about 3.2–3.8 pg per nucleus. The tetraploidization of the presumed 60-chromosome ancestor seems to have occurred at an early time of evolu- tion of the group. The divergence of the Acipenseridae into Scaphirhyninae and occurred without polyploidization. Within the Acipenser, polyploidization was one of the main genetic mecha- nisms of by which 8n and 16n-ploid were formed. Individual gene trees constructed for sequenced partial fragments of the 18S rRNA (230 base pairs, bp), 12S rRNA (185 bp), 16S rRNA (316 bp), and cytochrome b (270 bp) genes of two Eurasian (A. baerii and A. ruthenus) and two American (A. transmonta- nus and A. medirostris) species of Acipenser, Huso dauricus, Pseudoscaphirhynchus kaufmanni, Scaphir- hynchus albus, and Polyodon spathula showed a low level of resolution; the analysis of a combined set of data for the four genes, however, gave better resolution. Our phylogeny based on molecular analysis had two major departures from existing morphological hypotheses: Huso dauricus is a sister-species to Acipenser instead of being to all acipenseriforms, and Scaphirhynchus and Pseudoscaphirhynchus do not form a monophy- letic group. The constructed for the cytochrome b gene fragments (with inclusion of 7 addi- tional species of Acipenser) supported the conclusion that octoploid species appeared at least three times within Acipenser.

Introduction , and Teleostei; Bemis et al. 1997 this vol- ume). Most ichthyologists regard Polypteridae as Although a few species of Acipenser require revi- the of Acipenseriformes + sion, usually 24–25 extant sturgeon and two pad- (Patterson 1982). A comparison of partial sequenc- dlefish species, Polyodon spathula and Psephurus es of 28S rRNAs supported this relationship (Le et gladius, are included in the Acipenseriformes (Ro- al. 1993). In contrast to earlier works, Grande & Be- chard et al. 1991, Birstein 1993a). The extant mem- mis (1991) concluded that and stur- bers of this form the monophyletic sister- geons are sister taxa, and that extinct gen- group of all extant Neopterygii (e.g., Lepisosteidae, era such as Chondrosteus lie outside this .

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Within Acipenseriformes, all workers agree that ous occur in Central (Nesov & Kaznyshkin the Acipenseridae and Polyodontidae diverged pri- 1983). or the Late (Berg 1948a, Yakovlev 1977, The -western American group consists of 1986, Grande & Bemis 1991, Jin 1995, Grande & Be- Acipenser sinensis, A. dabryanus, A. medirostris, mis 1996, Bemis et al. 1997). Within the Acipenseri- and A. transmontanus; the external morphology of dae, the subfamily (Central the last two species is very similar (Findeis 1993). Asian species of Pseudoscaphirhynchus plus North All of these species seem to have common Tertiary American species of Scaphirhynchus) was tradi- roots (Artyukhin & Andronov 1990). The two ex- tionally considered the sister group of all other stur- tant paddlefish species also indicate a trans-Pacific geons (Berg 1905, Vasiliev 1985). Another interpre- pattern that is strengthened by the inclusion of fos- tation, based on osteology, was proposed by Findeis sil taxa, such as Crossopholis and Paleo- (1993, 1997 this volume), who found that Huso lacks cene (Grande & Bemis 1991, Jin many characters found in Acipenser, Scaphirhyn- 1995, Bemis et al. 1996, Grande & Bemis 1996). chus and Pseudoscaphirhynchus. On this basis, he The third group includes European and Amer- considered Scaphirhynchinae as a derived group ican Atlantic (Vladykov & Greeley 1963, within Acipenseridae (Findeis 1997). This point of Kinzelbach 1987, Holcˇı´k et al. 1989). Probably, the view was disputed since the description of the Pseu- European A. sturio has many primitive characters doscaphirynchus species 120 ago (Kessler of the genus (Nesov & Kaznyshkin 1977). Once con- 1877, Berg 1905), while other researchers consid- sidered a subspecies of A. sturio, the American At- ered Scaphirhyninae as the oldest group within Aci- lantic sturgeons were subsequently split off as a sep- penseridae (Bogdanov 1887). The examination of arate species, A. oxyrinchus (Vladykov & Greeley generic relationships within Acipenseriformes us- 1963). Then, two subspecies, A. o. oxyrinchus and ing molecular phylogenetic methods was one of the A. o. desotoi, were described within A. oxyrinchus main goals of this paper. (Vladykov 1955, Vladykov & Greeley 1963). The According to Nesov & Kaznyshkin (1977), extant origin and spread of the probably species of Acipenser belong to different evolution- reflect close Tertiary links between Europe and ary lineages which diverged long time ago. Artyuk- (Artyukhin & Andronov 1990). hin & Andronov (1990) and Artyukhin (1995) pro- The freshwater sturgeons of eastern North Amer- posed that there were at least four main regions in ica, belonging to the fourth group, the lake stur- which the speciation and spread of sturgeons took geon, A. fulvescens, and , A. bre- place: (1) the Ponto-Caspian area; (2) China-west- virostrum, are possibly closely related (Vladykov & ern America; (3) the Atlantic area; and (4) eastern Greeley 1963) and may have originated on the East- North America. The group of Ponto-Caspian spe- ern coast of North America (Artyukhin & Andro- cies includes most of the Eurasian species (Acipen- nov 1990). ser gueldenstaedtii, A. persicus, A. stellatus, A. ruth- Evidence for of these four groups re- enus, A. nudiventris, Huso huso, A. baerii; Berg mains uncertain, and the relationships among them 1948b, Holcˇı´k et al. 1989, Pirogovsky et al. 1989, are unknown. Recently, Artyukhin (1995) publish- Shubina et al. 1989, Vlasenko et al. 1989a, b, Soko- ed a first phylogenetic tree based on general data of lov & Vasilev 1989a–c, Ruban 1997 this volume), as morphology, biogeography and karyology (but not well as Amur River endemics (A. schrenckii and the DNA content) of Acipenser. Atryukhin’s Huso dauricus; Artyukhin 1994, Krykhtin & Svir- scheme is the first modern attempt to reconstruct skii 1997 this volume), and, possibly, an Adriatic relationships within the genus Acipenser (see Be- species A. naccarii (Tortonese 1989, Rossi et al. mis et al. 1997, for a history of the 19th century at- 1991). The origin of Ponto-Caspian species might tempts to subdivide Acipenser into subgenera). have been associated with brackish-water deriva- Artyukhin & Andropov (1990) wrote: ‘It is quite tives of the Tethys Sea. The oldest extinct forms of evident that methods of biochemical genetics and the Ponto-Caspian group from the Upper Cretace- karyology, as well as paleontological data and cur-

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Table 1. Chromosome numbers and DNA content in the Acipenseriformes, Lepisosteiformes, and Amiiformes1.

Species Chromosome DNA content in Ploidy, n Reference numbers pg (according to the DNA content)

Order Acipenseriformes Polyodontidae North America Polyodon spathula Tennesse River, 120 – – Dingerkus & Howell (1976) Kentucky – 3.92 4 Tiersch et al. (1989) – 4.892 4 Blacklidge & Bidwell (1993) Moscow , Russia – 3.172 4 Birstein et al. (1993) family Acipenseridae subfamily Acipenserinae Europe Acipenser gueldenstaedtii Volga River 250 ± 8 – – Birstein & Vasiliev (1987) 7.872 8 Birstein et al. (1993) 247 ± 8 – – Vasiliev (1985) Sea of Azov 250 ± 8 – – Vasiliev (1985), Arefjev (1989a) Italy, cell culture 256 ± 8 – – Fontana et al. (1995) A. naccarii Italy 239 ± 7 – – Fontana & Colombo (1974) 246 ± 8 – – Fontana (1994) Italy, cell culture 246 ± 8 – – Fontana et al. (1995) A. nudiventris Black Sea 118 ± 2 – – Arefjev (1983), Vasiliev (1985) , Uzbekistan (Central Asia) – 3.902 4 Birstein et al. (1993) A. persicus Caspian Sea > 200 – – Fashkhami (pers. comm.) A. ruthenus Volga River 118 ± 2 – – Vasiliev (1985), Birstein & Vasiliev (1987) – 3.742 4 Birstein et al. (1993) Don River 118 ± 3 – – Arefjev (1989b) Danube, Yugoslavia 116 ± 4 – – Fontana et al. (1977) Danube, Slovakia 118 ± 3 – – Rab (1986) Italy () 118 ± 4 – – Fontana (1994) Italy, cell culture 118 ± 9 – – Fontana et al. (1995) A. stellatus Volga River 118 ± 2 – – Birstein & Vasiliev (1987) – 3.742 4 Birstein et al. (1993) A. sturio Italy 116 ± 4 – – Fontana & Colombo (1974) – 3.64 4 Fontana (1976) – 3.26 4 Mirsky & Ris (1951) Huso huso Don River 118 ± 2 – – Serebryakova et al. (1983), Arefjev (1989b) Volga River 118 ± 2 – – Birstein & Vasiliev (1987) – 3.172 4 Birstein et al. (1993) Italy 116 ± 4 – – Fontana & Colombo (1974) – 3.64 4 Fontana (1976) Asia Siberia Acipenser baerii Lena River 248 ± 5 – – Vasiliev et al. (1980) Italy (aquaculture) 246 ± 8 Fontana (1994)

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Table 1. (Continued).

Species Chromosome DNA content in Ploidy, n Reference numbers pg (according to the DNA content)

Far East and China A. mikadoi (A. medirostris mikadoi) Tumnin (Datta) River [500?]3 14.202 16 Birstein et al. (1993) A. schrenckii Amur River [240?]5 – – Sereberyakova (1970) A. sinensis River 264 ± – – Yu et al. (1987) Huso dauricus Amur River [120?]5 – – Serebryakova (1969) – 3.782 4 Birstein et al. (1993) North America A. brevirostrum and South Carolina, USA [360? or 500?]3 13.082 12 (?) or 16 Blacklidge & Bidwell (1993) A. fulvescens [250?]3 8.902 8 Blacklidge & Bidwell (1993) A. medirostris Washington [250?]3 8.822 8 Blacklidge & Bidwell (1993) A. oxyrinchus desotoi Florida [120?]3 4.552 4 Blacklidge & Bidwell (1993) A.o. oxyrinchus Halifax, cell culture (99–112)7 – – Li et al. (1985) A. transmontanus San Francisco Bay, , cell culture (230 ±)8 – – Hedrick et al. (1991) – 9.552 8 Blacklidge & Bidwell (1993) Snake River, Idaho – 9.122 8 Blacklidge & Bidwell (1993) Columbia River, Washington – 9.592 8 Blacklidge & Bidwell (1993) Italy (aquaculture) 248 ± 8 – – Fontana (1994) subfamily Scaphirhynchinae Central Asia Pseudoscaphirhynchus kaufmanni Amu Darya River, Uzbekistan [120?]3 3.472 4 Birstein et al. (1993) North America Scaphirhynchus platorynchus 112 ± 3.62 4 Ohno et al. (1969) Order Lepisosteiformes family Lepisosteidae North America oculatus 68 ± 2.84 2 Ohno et al. (1969) L. osseus 56 – – Ojima & Yamano (1980) L. platostomus 54 – – Ueno (1985), cit. in Suzuki & Hirata (1991) Order family Amiidae North America calva 46 ± 2.44 2 Ohno et al. (1969) 2.36 2 Mirsky & Ris (1951) 46 2.04 2 Suzuki & Hirata (1991)

1 All species investigated karyologically so far are listed; the DNA content values for all species studied are given. 2 Determined by flow cytometry. 3 Chromosome number is assumed on the basis of the DNA content. 4 Determined by microdensitometry of Feulgen-stained nuclei. 5 Only macrochromosomes were counted precisely. 6 Determined by the biochemical Schmidt-Thankauser method. 7 Chromosome number was determined in cardiac tissue cell culture. 8 Chromosome number was determined in cell cultures: the modal 2n in a spleen cell line was 219, and in a heart cell line, 237–243 (Hedrick et al. 1991).

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Table 2. Characteristics of karyotypes of several acipenseriform species1.

Species Chromo- Number of Number of Number of Approximate Reference some large meta- large micro- arm number, number centrics plus telocentrics chromo- NF medium meta/ somes submeta- centrics, M + m/sm

1. Tetraploid species Family Polyodontidae Polyodon spathula 120 8 + 36 4 72 164 Dingerkus & Howell (1976) Family Acipenseridae Acipenser nudiventris Black Sea, Russia 118 ± 3 8 + 46 4 60 ± 3 172 Arefjev (1983) A. ruthenus Sea of Azov, Russia 118 ± 3 8 + 50 4 56 ± 3 176 Arefjev (1989a) Volga River, Russia 118 ± 2 8 + 50 4 56 ±23 176 Birstein & Vasiliev (1987) Danube, Slovakia 118 ± 4 8 + 50 4 56 ± 4 176 Rab (1986) Danube, Yugoslavia 116 ± 2 8 + 48 4 56 + 4 170 Fontana et al. (1977) A. stellatus Volga River, Russia 118 ± 2 8 + 48 4 58 ± 2 174 Birstein & Vasiliev (1987) A. sturio Italy 116 ± 4 8 + 48 4 56 ± 4 172 Fontana & Colombo (1974) Huso huso Volga River, Russia 118 ± 2 8 + 54 4 52 ± 2 180 Birstein & Vasiliev (1987), Arefiev & Nikolaev (1991) Sea of Azov, Russia 118 ± 3 8 + 54 4 52 ± 3 180 Serebryakova et al. (1983), Arefiev (1989b) Italy 116 ± 4 8 + 52 4 52 ± 4 176 Fontana & Colombo (1974) Scaphirhynchus platorynchus USA 112 ± 2 8 + 52 4 48 ± 172 Ohno et al. (1969) 2. Octoploid species Family Acipenseridae Acipenser baerii Lena River, Siberia, Russia 248 ± 5 16 + 42 190 308 Vasiliev et al. (1980) A. gueldenstaedtii Volga River, Russia 250 ± 8 16 + 76 155 339 Birstein & Vasiliev (1987) Sea of Arzov, Russia 250 ± 8 16 + 82 152 348 Arefjev (1989a) A. naccarii Italy (wild) 239 ± 7 16 + 76 147 331 Fontana & Colombo (1974) Italy, aquaculture 241 ± 3 16 + 74 151 331 Arlati et al. (1995) A. sinensis China 264 ± 3 16 (?) + 82 166 362 Yu et al. (1987)

1 A revision of data from papers mentioned as References. The numbers of m/sm, microchromosomes, and NF are given approximately, since it is impossible to discriminate the form and exact number of small chromosomes and microchromosomes. For octoploid species, the number of telocentrics and microchromosomes is given together because there is no clear size difference between these two kinds of chromosomes. Usually there are 2–5 middle and/or small-sized telocentric pairs in these karyotypes.

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110969 EBFI ART.NO 1621 (349) ORD.NO 231349.F 132 rent views on paleogeography will provide useful The size of macrochromosomes in both groups is tools for resolving complex relationships and phy- between 2–5 µm, and the majority of macrohromo- logeny of sturgeons’. In the first part of this paper somes are the meta- and submetacentrics (Table 2). we review all cytogenetic data available on Acipen- One third to one half of the chromosome number in seriformes and make some new conclusions rele- both groups is comprised of microchromosomes of vant to the four groups within Acipenser mentioned a very small size (about 1 µm). Karyotypes of the above. In the second part we describe experimental 120-chromosome species typically consist of 4 pairs data on the molecular phylogeny of Acipenseri- of large metacentrics (no. 1–4), 5 pairs of large but formes. Because multiple gene regions have been somewhat smaller metacentrics (no. 5–9), about 20 useful in other groups of fishes (reviews in Normark pairs of medium-sized metacentrics and/or sub- et al. 1991, Stock et al. 1991a, Meyer 1993, Patterson metacentrics of gradually decreasing size (no. 10– et al. 1993), we believed that they might also provide 30), one pair of comparatively large telocentrics reasonable character state information for acipen- (no. 30), one pair of small telocentrics, and approxi- seriforms. Consequently we amplified and se- mately 56 ± 4 microchromosomes of different form quenced partial fragments of 18S rRNA, 12S rRNA, (Table 2). The difference between karyotypes of 16S rRNA, and cytochrome b genes of a few repre- representatives of two lineages of the extant aci- sentatives of all lineages of this order. We included penseriforms, the Polyodontidae (Polyodon spath- the phylogenetic analysis of the combined molecul- ula) and Acipenseridae (all other 120-chromosome ar and morphological data for all the species we species in Table 1), as well as of the lineages within studied. Also, we examined relationships among the Acipenseridae (Huso huso, the 120-chromo- representatives of four species groups of the genus some species of the genus Acipenser, and Scaphir- Acipenser recognized by Artyukhin (1995), using hynchus platorynchus), seems to be small. Evident- data for a partial sequence of the cytochrome b ly, the ancestral acipenseriform karyotype was pre- gene. served in these fishes without dramatic changes during diverisification of the group. In general, few karyotypic changes are noticeable Acipenseriform cytogenetics: an overview among the species of Acipenser with 120-chromo- somes (Table 2). The karyotype of Huso huso is Main karyotypic characteristics, DNA content, and more symmetric than those of the 120-chromosome species of Acipenser, i.e., it contains more biarmed chromosomes and fewer microchromosomes (see Karyotypes of about half of all sturgeon species Morescalchi 1973). Also, there is a small difference have been described and the DNA content in most among the 120-chromosome species in the size of a sturgeon species and has been pair of large telocentrics (no. 30): it is small in A. measured (Table 1). Acipenseriform karyotypes in- sturio (Fontana & Colombo 1974) and A. nudiven- vestigated so far have two particular characteristics: tris (Arefjev 1983) and it is as large as pair no. 8 or 9 (1) they are large; and (2) they consist of macro- and in Huso huso (Fontana & Colombo 1974, Birstein & microchromosomes. According to the number of Vasiliev 1987, Arefjev 1989b) and A. ruthenus (Rab chromosomes (2n), the species can be divided into 1986, Birstein & Vasiliev 1987). two groups: those with about 120 chromosomes The similarity of karyotypes of the 120-chromo- (e.g., Huso huso, H. dauricus, Acipenser ruthenus, some acipenseriforms points to a generally slow A. stellatus, A. nudiventris, A. sturio, and Polyodon rate of karyological evolution. This correlates with spathula), and those with 240 chromosomes (e.g., A. a slow rate of nuclear DNA evolution: practically all gueldenstaedtii, A. naccarii, A. baerii, A. schrenckii, genome fractions (both the repeated and unique se- and A. transmontanus). By comparison to the 120- quences) are homologous in Acipenser ruthenus, A. chromosome species, the 240-chromosome species stellatus, A. gueldenstaedtii, and H. huso, and the are tetraploids. number of nucleotide substitutions in the first frac-

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110969 EBFI ART.NO 1621 (349) ORD.NO 231349.F 133 tion is 0–2.65, and 1.5–2.7% in the second (Kedrova chromosome number in three generations of the et al. 1980). These data were supported by our re- ‘bester’, the fertile between , Huso sults from sequencing the 18S genes, which are al- huso, and , A. ruthenus, does not differ from most invariable among acipenseriforms (see be- that of parental species, and a gradual displacement low). of karyotypic parameters (numbers of bi- and uni- A low degree of protein evolution is usually char- armed chromosomes) towards those of the sterlet acteristic of the acipenseriforms, especially the 120- occurs (Arefjev 1989b). The sterlet has evidently chromosome species. A mean heterozygosity for not only invariable DNA content, but also a dom- three freshwater species, Polyodon spathula, Sca- inant karyotype. The situation is very unusual, be- phirhynchus platorynchus and S. albus, is between cause, as a rule, the karyotypes of interspecies 0.010 and 0.017 (Carlson et al. 1982, Phelps & Allen- hybrids are more variable than karyotypes of the dorf 1983) and is the highest (of all species investi- parental species (Arefjev 1991, Arefjev & Filippova gated) in the anadromous Acipenser stellatus, 0.093 1993). (Ryabova & Kutergina 1990). All of these species The Asian green (Sakhalin) sturgeon, A. mikadoi are 120-chromosome forms. The mean heterozy- (or A. medirostris mikadoi as explained below), and gosity for other osteichthyans is 0.051 (Ward et al. the American shortnose sturgeon, A. brevirostrum, 1992). The complete lack of genetic divergence at have even higher DNA contents than the 240-chro- the protein level between the two species of Sca- mosome forms. The DNA content of A. mikadoi is phirhynchus is very unusual for fishes, especially 14.2 pg per nucleus, four times higher than in the because freshwater fishes typically exhibit subpop- 120-chromosome species or roughly twice that of ulational differentiation significantly higher than the 240-chromosome forms (Birstein et al. 1993). In that of anadromous and especially marine species American , A. medirostris, it is about (Ward et al. 1994). half of this value (8.8 pg, Blacklidge & Bidwell The DNA content (2C) of all of the 120-chromo- 1993). Therefore, the American green sturgeon some species is about 3.7–3.9 pg (in Polyodon spath- seems to be a typical 240-chromosome form, while ula it is a little lower, 3.2 pg), whereas in the 240- the might be predicted to have chromosome species it is twice as high, 7.9–8.3 pg twice the number of chromosomes, or around 500. (Birstein et al. 1993, Table 1). Moreover, the same If so, the Sakhalin sturgeon would have the highest tendency in DNA content is in American diploid number reported in . But pol- sturgeons whose karyotypes have not been investi- yploidization can occur without an increase in chro- gated: A. oxyrinchus desotoi (2C = 4.6 pg) is evi- mosomes number, as in several species of dently a 120-chromosome subspecies, whereas A. (see below), and only direct karyotypic study can fulvescens and A. medirostris (American form) are address the chromosome number and morphology 240-chromosome species, 2C = 8.8–8.9 pg (Black- of the Sakhalin sturgeon. DNA content data sup- lidge & Bidwell 1993; the slightly higher DNA con- port the old point of view that the Asian form of A. tent for all American species as compared with the medirostris is a separate species, A. mikadoi (Hil- Eurasian species in Table 1 is due to the different gendorf, 1892), or a subspecies, A. medirostris mika- methods used by Birstein et al. 1993, and Blacklidge doi (Shmidt 1950, Lindberg & Legeza 1965; in Table & Bidwell 1993). DNA content in Pseudoscaphi- 1 it is mentioned as a species, see Birstein 1993b). rhynchus kaufmanni (3.2 pg) is slightly lower than in Although Blacklige & Bidwell (1993) consider A. the sterlet, A. ruthenus (3.7 pg), which was used by brevirostrum to be an allopolyploid (12n = 360), a Birstein et al. (1993) as a standard species for com- descendant of ancestral spontaneous triploids, allo- parative measurments due to its invariable DNA polyploidy is unknown in other acipenserids, and it content. However, this difference is so small that we is more logical to propose that this is a 16n-ploid. predict P. kaufmanni is a 120-chromosome species. An investigation of active nucleoli gave addition- The sterlet, Acipenser ruthenus, has other prop- al information about ploidy in the acipenseriforms. erties which attest to its genetic stability. The basic There are different modal numbers of nucleoli per

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110969 EBFI ART.NO 1621 (349) ORD.NO 231349.F 134 nucleus in species investigated (Table 3): 2–4 (maxi- of four homologs, and Dingerkus & Howell (1976) mum 6) in the 120-chromosome species, and 6–8 divided the karyotype of 120-chromosome Polyo- (maximum 11–12) in the 240-chromosome species of don spathula into 30 groups consisting of four chro- Acipenser (Birstein & Vasiliev 1987, Fontana 1994). mosomes of similar morphology. If it is taken into In A. ruthenus, 4n, the NORs are located in two consideration that this species has 4 active nucleoli pairs of small chromosomes, a pair of metacentrics per nucleus (see above), it is clear that P. spathula is and a pair of telocentrics (possibly no. 30; Rab 1986, a tetraploid. The same procedure of chromosome Birstein & Vasiliev 1987, Fontana 1994). According grouping (but with greater uncertainity) can be to Arefjev (1993), in octoploid A. baerii NORs are done for A. ruthenus and H. huso (Birstein & Vasi- located also on two pairs of chromosomes, while liev 1987). From this comparison, one can conclude Fontana (1994) found two quadruplets bearing that the 120-chromosome species are in reality tet- NORs in A. baerii, A. naccarii, and A. transmonta- raploids, while the 240-chromosome species are re- nus. The average number of nucleoli in Polyodon ally octoploids, and the ploidy of A. mikadoi and A. spathula is 4 (Dingerkus & Howell 1976), as many as brevirostrum possibly is 16n. in the 120-chromosome species of Acipenser (Table Further evidence that the 120-chromosome spe- 3). Usually the number of nucleoli in diploid tele- cies are tetraploids comes from the existence of du- osts is half that of the 120-chromosome acipenser- plicated loci, a common characteristic of pol- forms and equals 1–2 nucleoli per nucleus (review in yploids. A high level of duplicated loci (31%) was Birstein 1987). found in A. stellatus (Nikanorov et al. 1985, Ryabo- The tendency seen in 120-chromosome sturgeons va & Kutergina 1990); duplicated loci were also to have more nucleoli than in diploid is ap- found in Huso huso (Slynko 1976). In A. stellatus, parently caused by their high ploidy. Ohno et al. duplicated loci Ldh3 and Ldh4 are located at two (1969) arranged the first 64 chromosomes of Sca- different chromosomes (Kutergina & Ryabova phirhynchus platorynchus, 4n=116± into 16 groups 1990). In Polyodon spathula the expression of dupli-

Table 3. Number and location of nucleolar organizer regions (NORs) in Acipenseriformes (data on Ag-staining).

Species Chromosome Number of NORs Number of NORs-bearing Reference number per nuclei1 chromosomes and NORs’ location2

Family Polyodontidae Polyodon spathula 120 4 Dingerkus & Howell (1976) Family Acipenseridae 1. Tetraploid species Acipenser ruthenus 118 2–3 (1–6) Two pairs (T & m), telomeric Birstein & Vasiliev (1987) 118 – Two pairs, telomeric Fontana (1994) A. stellatus 118 2–3 (1–6) Two pairs (both (?) m), telomeric Birstein & Vasiliev (1987) Huso huso 118 2–3 (1–6) Two pairs (both (?) m), telomeric Birstein & Vasiliev (1987) 2. Octoploid species Acipenser baerii 250 4 (2–6) Two pairs (T & m) Arefjev (1993) 250 – Two quadruplets Fontana (1994) A. gueldenstaedtii 250 6–8 (2–12) – Birstein & Vasiliev (1987) A. naccarii 246 – Two quadruplets Fontana (1994) A. transmontanus 248 – Two quadruplets Fontana (1994)

1 The modal number; a variation in the number is given in the parenthesis. 2 T means medium-sized telocentric, and m, microchromosome; telomeric means telomeric location of NORs.

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Table 4. Natural hybridization of sturgeon species and their ploidy.

Interspecies hybrids Intergenera hybrids

1. Caspian Sea basin1 (a) Volga River A. ruthenus (4n) × A. stellatus (4n) H. huso (4n) × A. gueldenstaedtii (8n) A. stellatus (4n) × A. ruthenus (4n) H. huso (4n) × A. ruthenus (4n) A. nudiventris (4n) × A. gueldenstaedtii (8n) A. gueldenstaedtii (8n) × A. ruthenus (4n)2 A. gueldenstaedtii (8n) × A. stellatus (4n) A. gueldenstaedtii (8n) × A. persicus (8n)3 (b) Kama River Huso huso (4n) × A. nudiventris (4n) H. huso (4n) × A. gueldenstaedtii (8n) H. huso (4n) × A. stellatus (4n) A. ruthenus (4n) × H. huso (4n) (c) Ural River A. nudiventris (4n) × A. stellatus (4n) H. huso (4n) × A. stellatus (4n) A. stellatus (4n) × A. nudiventris (4n) (d) Kura River A. nudiventris (4n) × A. stellatus (4n) H. huso (4n) × A. nudiventris (4n) A. stellatus (4n) × A. nudiventris (4n) A. nudiventris (4n) × A. gueldenstaedtii (8n) (e) Sefir-Rud River A. nudiventris (4n) × A. stellatus (4n) A. nudiventris (4n) × A. gueldenstaedtii (8n) (f) Caspian Sea H. huso (4n) × A. persicus (8n) 2. Sea of Azov basin4 Don River A. ruthenus (4n) × A. stellatus (4n) 3. Black Sea basin5 (a) Danube A. ruthenus (4n) × A. stellatus (4n) A. gueldenstaedtii (8n) H. huso (4n) A. ruthenus (4n) × A. nudiventris (4n) A. stellatus (4n) × H. huso (4n) A. stellatus (4n) × A. ruthenus (4n) A. nudiventris (4n) × H. huso (4n) A. ruthenus (4n) × A. gueldenstaedtii (8n) H. huso (4n) × A. stellatus (4n) A. stellatus (4n) × A. gueldenstaedtii (8n) A. nudiventris (4n) × A. gueldenstaedtii (8n) A. sturio (4n) × A. gueldenstaedtii (8n) (b) Black Sea A. gueldenstaedtii (8n) × A. sturio (4n) A. gueldenstaedtii (8n) × A. nudiventris (4n) 4. Siberian rivers Main rivers (Yenisey, Lena, Ob, Kolyma)6 Amur River7 A. baerii (8n) × A. ruthenus (4n) Huso dauricus (4n) × A. schrenckii (8n?) 5. Central Asia8 Amu-Darya River Pseudoscaphirhyncus kaufmanni (4n) × P. hermanni 6. North America9 Missouri and Rivers Scaphirhynchus albus (?) × S. platorynchus (4n)

1 Data from Berg (1911, 1948b), Kozhin (1964), Kozlov (1970), Legeza (1971), and Keyvanfar (1988). 2 In the early 1950s, this hybrid was the most numerous (46% of all hybrids caught; Konstantinov et al. 1952). 3 Data from Vlasenko et al. (1989b). 4 Data from Berg (1948b) and Kozhin (1964). 5 Data from Antipa (1909), Antoni-Murgoci (1946), Banarescu (1964), and Berg (1948b). 6 Data from Berg (1948b). 7 Data from Berg (1948b), Wei et al. (1996), and Krykhtin & Svirskii (1996). 8 Data from Nikolskii (1938) and Berg (1948b). 9 Data from Carlson et al. (1985).

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110969 EBFI ART.NO 1621 (349) ORD.NO 231349.F 136 cated loci is much lower, only 6% (Carlson et al. characters studied, 9 deviated toward the maternal 1982). Why this is so is unknown. In this species du- species (beluga), and 18 deviated toward the pater- plicated loci for insulin, glucagon and glucagon-like nal species (sterlet) (Krylova 1981). The meristic peptide were found (Nguyen et al. 1994). characters (the number of dorsal, lateral, and ven- As for the 240-chromosome octoploid species, it tral scutes) deviated toward the maternal species is evident that two forms of vitellogenin monomers (beluga). The ease of hybridization of sturgeon spe- in American A. transmontanus (Bidwell et al. 1992) cies allowed to show the maternal inheritance of and two forms of growth hormones in Russian A. some behavior characters of sturgeons (Marshin et gueldenstaedtii (Yasuda et al. 1992) are a result of al. 1969). polyploidization. A higher ploidy level seems to be a reason for a higher heterozygosity in A. guelden- staedtii compared to the 120-chromosome species of Problem of the ancestral karyotype Acipenser from the same geographic area (Slynko 1976, Keyvanfar 1988, Kuzmin 1991). Possibly, a Data presented above support the hypothesis of the high level of ploidy causes a high variation in the tetraploid origin of 120-chromosome acipenseri- mean heterozygosity of American octoploid A. forms from a 60-chromosome ancestor before the transmontanus (0.014–0.069, Bartley et al. 1985). radiation of this order (Dingerkus & Howell 1976, Hemoglobin, the only protein examined in the 16n- Carlson et al. 1982). The karyotypes of Lepisostei- ploid A. mikadoi, is more heterogeneous (11 elec- dae () and Amiidae () are relevant to trophoretic fractions) than in tetra- (8–9 fractions) understanding the proposed acipenseriform ances- or octoploid (7–8 fractions) species of Acipenser tral karyotype. Gars have approximately 60 chro- (Lukyanenko & Lukyanenko 1994). mosomes (Table 1), but it seems that many karyo- Polyploidization is a relatively uncommon genet- logical changes have occurred during their evolu- ic mechanism in vertebrates, occurring only in lam- tion. The karyotype of Lepisosteus oculatus, 2n = preys, elasmobranchs, acipenseriforms, some 68, consists of many meta- and acrocentric macro- groups of teleosts (salmonids, cyprinids, and catos- chromosomes, as well as many microchromosomes tomids), (anurans), and lizards (review (Ohno et al. 1969), while the karyotype of L. osseus, in Birstein 1987). To date, polyploidization is un- 2n = 56, lacks microchromosomes (Ojima & Yama- known in or . It seems that the pol- no 1980). The karyotype of Amia calva is even more yploid state, and karyotypic and genomic similarity reduced, 2n = 46, but it still includes microchromo- of different acipenseriforms contribute to easy in- somes (Ohno et al. 1969, Suzuki & Hirata 1991). The terspecific and even intergeneric hybridization cellular DNA content of gars and Amia ranges from within the Acipenseridae. 2.0 to 2.8 pg per nucleus, which is approximately The Acipenseridae is the only group among ver- half that of the 120-chromosome acipenseriforms. tebrates all members of which can hybridize with Therefore, it is quite possible that the common an- each other in the wild if their spawning grounds cestor of acipenseriforms and neopterygians had a overlap (Table 4). The unique easiness of hybrid- karyotype of about 60 chromosomes consisting of ization of acipenserids was described by Russian micro- and macrochromosomes, with a DNA con- ichthyologists more than 100 years ago (Ovsyanni- tent about 2.0 pg per nucleus. kov 1870, Zograf 1887). Some hybrids (such as the Extant polypterids, which are the living members artificially obtained ‘bester’, H. huso × A. ruthenus, of the basal actinopterygian group , have and its reciprocal hybrid, Nikolyukin 1970), have 36 bi-armed chromosomes (except the desirable characteristics of fast growth and high weekesii, 2n = 38; reviews in Vervoort 1980, Suzuki viability, are fertile (which is also unusual for ver- et al. 1988, 1989). The DNA content in polypterids is tebrate hybrids) and are widely used in aquaculture considerably higher than in the acipenseriforms, 2C (Williot et al. 1993). Besters inherit a phenotype in- = 12–13 pg per nucleus (Vervoort 1980). It is evident termediate between the parental species. Of 29 that the polypterids are a cytogenetically advanced

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110969 EBFI ART.NO 1621 (349) ORD.NO 231349.F 137 group. Molecular data also support this conclusion: teristics of the karyotypes of spotted ratfish, gars, the 18S rRNA sequences in the species of the gen- and sturgeons, Ohno (1970) and later Dingerkus era Polypterus and Erpetoichthys are very similar to (1979) proposed that the ancestral karyotype of each other, but both are highly divergent from those gnathostomes consisted of approximately 50–60 of other gnathostomes (Stock et al. 1991a). macro- and microchromosomes. The karyotypes of chondrichthyans are more in- Recently a karyotype consisting of macro- and formative. Chondrichthyans are mostly tetraploids, microchromosomes was described in another living 4n = 90–104 (reviews in Schwartz & Maddock 1986, fish, the coelacanth chalumnae Asahida et al. 1988, 1993, Asahida & Ida 1989, 1990, (Bogart et al. 1994). It appears to include 16 pairs of Stingo et al. 1989, Stingo & Rocco 1991), and karyo- macro- and eight pairs of microchromosomes. This types contain macro- and microchromosomes. Ac- karyotype resembles to a high extent that of one of cording to cellular DNA content and DNA re-asso- the most primitive living frogs, Ascaphus truei, but ciation kinetics data (Olmo et al. 1982, Ida et al. this resemblance could be coincidental. 1985), the ploidy level of a few species of sharks is Karyotypes of acipenseriforms generally resem- higher, and in these cases polyploidization occurred ble karyotypes of primitive amphibians, not anu- without a change in the chromosome number (phe- rans as in L. chalumnae, but, instead, urodeles be- nomenon known as cryptopolyploidy; Wagner et al. longing to the family Hynobiidae. With several ex- 1993). The only extant chondrichthyan which pos- clusions, the karyotypes of hynobiids, 2n = 56–62, sibly retains a diploid karyotype is the spotted rat- consist of a few large and middle-sized pairs of bi- fish, Hydrolagus colliei: it has the lowest chromo- armed macrochromosomes, a few pairs of telocen- some number among elasmobranchs, 2n = 58, and tric macrochromosomes, and 15–20 pairs of micro- the lowest DNA content, 2C = 3.0 pg (Ohno et al. chromosomes (Morescalchi et al. 1979, King 1990, 1969). But this could be a derived condition, for in- Kohno et al. 1991). It seems that the ancestral karyo- sufficient taxa have been studied to draw any con- of these amphibians consisted of 60 macro- and clusion. Moreover, changes in DNA content occur microchromosomes. Because the karyotypes of hy- even during ontogenesis of this species: about 10% nobiids, as well as those of acipenseriforms, include of the genome is eliminated in somatic tissues as a large number of bi-armed macrochromosomes, compared with the germ cells (Stanely et al. 1984). they should be considered derived (as compared, In contrast to Acipenseriformes, the reduction of for instance, with those of the most ancient forms of chromosome number through fusion of micro- and chondrichthyans). small chromosomes into macrochromosomes was the main evolutionary karyotypic trend in chon- drichthyans (Schwartz & Maddock 1986, Stingo et Cytogenetic data and phylogeny of the Acipenseri- al. 1989). As a result, the karyotypes of advanced formes chondrichthyans consist of a smaller number of mainly bi-armed chromosomes, 2n = 50–70. An- It is impossible to infer generic interrelationships other considerable difference is that acipenseri- within the Acipenseridae from cytogenetic data. forms have numerous bi-armed macrochromo- Divergence of the three lines within the family oc- somes, whereas the most generalized, plesiomor- curred without polyploidization, and the ancestors phic elasmobranchs have only 2–3 pairs of bi-armed of all three lineages within the acipenserids seem to macrochromosomes, and up to 50 pairs represented have been tetraploids, 4n = 120. If the genus Huso by small telocentrics or by microchromosomes originated as the first outshoot within the Acipen- (Schwartz & Maddock 1986, Stingo & Rocco 1991). seridae (as proposed by Findeis 1993, 1997), then Moreover, the average DNA content in elasmo- this event was not accompanied by a substantial ka- branchs is much higher than in acipenseriforms (re- ryotypic change. The ancestral karyotype seems to views in Schwartz & Maddock 1986, Birstein 1987, have been retained without significant modifica- Asahida et al. 1988, 1993). Based on general charac- tion, since the karyotype of Huso huso is only slight-

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110969 EBFI ART.NO 1621 (349) ORD.NO 231349.F 138

Figure 1. A schematic representation of changes in ploidy in Acipenseriformes. M = macro-, m = microchromosomes. ly more symmetric than karyotypes of other acipen- content data) and 16n-ploid (according to DNA serids. A schematic course of possible cytogenetic content data) species. The octoploid 240-chromo- evolution within the Acipenseriformes is presented some sturgeon species seem to have originated in- in Figure 1. dependently in different regions. The closely relat- Cytogenetic data are helpful for understanding ed A. gueldenstaedtii and A. persicus may have a some relationships within the genus Acipenser, common origin and a common octoploid ancestor. where polyploidization was one of the main genetic Molecular data point to the close relatedness of A. mechanisms of speciation. The diversification of gueldenstaedtii to A. baerii (see below). this genus was accompanied by an appearance of Acipenser medirostris and A. transmontanus octoploid (according to the karyotypic and DNA have many similar characteristics in morphology,

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110969 EBFI ART.NO 1621 (349) ORD.NO 231349.F 139 biology, and ecology, as well as overlapping ranges character in cladistic terms) to the hypothetical in western North America (Hart 1973, Scott & grouping of species within the genus Acipenser Grossman 1973). Moreover, according to Artyuk- based on the species morphology, ecology, biogeog- hin & Andronov (1990), some ecological and bio- raphy and possible area of origin (Artyukhin & An- logical characteristics are common to these two spe- dronov 1990, Artyukhin 1994, 1995). cies and the , A. sinensis. All three Among fishes, an analogous mode of speciation species are octoploid (Table 1). Possibly, tetraploi- through multiple independent tetraploidization dization occurred in the ancestor of all three spe- events is characteristic of only one group of teleosts, cies. If the difference between the karyotype of A. the family (review in Buth et al. 1991). A sinensis and A. transmontanus (8n = 264 ± 3 and 230 tetraploid ancestor of two species, carpio ± or 248 ± 8, respectively is real, then it means that and auratus, appeared through tetraploi- at least two polyploidization events took place in dization about 16–20 million years ago (Risinger & the ancestral 120-chromosome form. The next step Larhammar 1993, Larhammar & Risinger 1994). of polyploidization which occurred in the 240-chro- Hexaploids in and (reviews in Vasi- mosome ancestor of A. medirostris and A. mikadoi, liev 1985, Buth et al. 1991, Golubtsov & Krysanov resulted in the formation of the genome of A. mika- 1993), as well as a 16/20n-ploid Asian species Dip- doi (which is, therefore, the youngest in this group tychus dipogon (Yu & Yu 1990) also were formed of species). The Amur River sturgeon, A. schren- within Cyprinidae. Moreover, as a result of a tetra- ckii, also seems to be an octoploid (but these data ploidization event which occurred approximately are preliminary, see Table 1), as the species of the 50 million years ago an ancestor of another family, trans-Pacific A. sinensis-A. medirostris-A. trans- , appeared within this group (Uyeno montanus group, and lives in a close geographic & Smith 1972). Catostomids are considered to have area. According to Artyukhin (1994, 1995), A. been tetraploid since then (Ferris & Whitt 1979, Ue- schrenckii is closely related to the Ponto-Caspian no et al. 1988, Tsoi et al. 1989). Tetraploids were also species A. nudiventris and A. ruthenus. formed in another family closely related to Cyprini- It is difficult to draw any conclusions concerning dae, the (review in Vasiliev 1985). Only the ancestor of A. brevirostrum, the species with the one other group of teleosts, the (which second highest level of DNA content. The other includes three subfamilies, Coregoninae, Thymalli- species of the Eastern Coast of North America, A. nae, and Salmoninae, sensu Nelson 1994) also orig- oxyrinchus, is closely related to the European 120- inated from a tetraploid ancestor (Gold 1979, Al- chromosome Atlantic sturgeon, A. sturio, and, ac- lendorf & Thorgaard 1984). By contrast with the cording to the DNA content, has the same ploidy acipenseriforms, cyprinids, and catostomids, a de- (Table 1). Possibly, A. brevirostrum is also related to crease in chromosome number through centromer- some European sturgeons (A. nudiventris; see be- ic fusion was characteristic for different lineages of low), and originated from these European-related salmonids (Vasiliev 1985, Buth et al. 1991). There- ancestors through polyploidization. Because of its fore, a gradual increase in chromosome number high ploidy level, A. brevirostrum might be a young through polyploidization has occurred a few times species among East American representatives of during the history of actinopterygians. the genus Acipenser. As for another American spe- cies, the freshwater , A. fulvescens, which is also an octoploid like A. oxyrinchus (ac- Molecular phylogeny of the Acipenseriformes cording to its DNA content), its origin and relation- ships with the other species are still unclear, al- Our first objective was to search for molecular syn- though it is morhologically similar to A. breviros- apomorphies of Acipenseriformes and its major in- rum (Vladykov & Greeley 1963, Findeis 1993). cluded using representatives of all extant Therefore, the cytogenetic data provide additional genera (except the , Psephurus information (ploidy level can be considered as a gladius, due to our inability to obtain suitable tis-

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110969 EBFI ART.NO 1621 (349) ORD.NO 231349.F 140 sue). Our data were polarized using other Actinop- found (Brown et al. 1992b, 1993). Fifty percent of A. terygii, Polypterus and Amia. We also attempted a medirostris studied were also heteroplasmic; D- synthesis of morphological, karyological and mole- loops of these individuals included from one to four cular characters as related to relationships among repeats (Brown et al. 1996). The average size of Acipenseriformes. Finally, we examined interrela- mtDNA of the lake stugeon, A. fulvescens, is ap- tionships among representatives of Artyukhins spe- proximately the same as that of , cies groups proposed for Acipenser (see above). 16.6–16.9 kb (Gue´nette et al. 1993, Ferguson et al. Whereas our examination of acipenseriform taxa 1993) or 16.1–16.5 kb (Brown et al. 1996). No hetero- concentrated on comparisons of species belonging plasmy was detected in A. fulvescens and A. oxyrin- to different genera or species within the genus Aci- chus (Brown et al. 1996). All individuals of A. ful- penser, most previous workers have concentrated vescens studied had one of five possible mtDNA on intraspecies structure using the control region size variants which closely corresponded to A. (D-loop) of the mtDNA. Buroker et al. (1990) transmontanus with one to five repeat units. In A. showed that in the American white sturgeon, Aci- oxyrinchus, nearly every individual was fixed for penser transmontanus, mtDNA size varies between mtDNA roughly equivalent in size to the smallest 16.1 and 16.7 kb depending on the number of tan- repeat found in the other species. Restriction analy- demly repeated 82 nucleotide sequences in the con- sis of mtDNA (Bowen & Avise 1990, Avise 1992) trol region of the mtDNA. Nearly 50% of the indi- and partial sequencing of the control region (Mira- viduals studied by Brown et al. (1992a) were hetero- cle & Campton 1995, Ong et al. 1996, Wirgin et al. plasmic (i.e., had multiple copies of different 1997 this volume) were used for inferring relation- mtDNA types within an individual) for length vari- ships between subspecies and populations of A. oxy- ation, with six different mtDNA length variants

Table 5. List of sturgeon species and blood samples studied.

Species Species Geographical region Number of blood (or Collector number tissue) samples

1. Acipenser baerii1 Lena River, Siberia, Russia (Moscow Aquarium) 2 V. Birstein 2. A. brevirostrum Connecticut River, MA, USA () B. Kynard 3. A. gueldenstaedtii1 Volga River, Russia (Moscow Aquarium) 2 V. Birstein 4. A. medirostris Columbia River, OR, USA 1 J. North 5. A. mikadoi Tumnin River, Russia 2 (fragments of muscles) E. Artuykhin 6. A. naccarii Ferrara, Italy (Aquarium) 2 F. Fontana 7. A. nudiventris1 Aral Sea, Uzbekistan (Moscow Aquarium) 2 V. Birstein 8. A. oxyrinchus oxyrinchus Hudson River 2 (fragments of muscles) J. Waldman 9. A. ruthenus1 Volga River, Russia (Moscow Aquarium) 2 V. Birstein 10. A. stellatus1 Volga River, Russia (Moscow Aquarium) 2 V. Birstein 11. A. transmontanus Columbia River, OR, USA 2 J. North 12. Huso dauricus1 Amur River, Siberia, Russia (Moscow Aquarium) 2 V. Birstein 13. Pseudoscaphirhynchus kaufmanni1 Amu-Darya River, Uzbekistan (Moscow Aquarium) 2 V. Birstein 14. Scaphirhynchus albus Yellowstone River, MT, USA 2 H. Bollig 15. Polyodon spathula1 Moscow Aquarium 1 V. Birstein

1 These samples were used for the DNA content measurements in Birstein et al. (1993); see Table 1 above.

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110969 EBFI ART.NO 1621 (349) ORD.NO 231349.F 141 rinchus. Both subspecies, A. o. oxyrinchus and A. individuals used for DNA content measurements oxyrinchus desotoi, exhibited low mtDNA diversity. by Birstein et al. (1993). Also, we isolated DNA The order and transcriptional polarity of three from alcohol-fixed samples of muscles of Amia cal- mitochondrial genes in A. transmontanus (genes for va and provided by Paul Vra- cytochrome b, threonine and proline tRNAs) are na (American Museum of Natural History, New identical to those of other vertebrates (Gilbert et al. York). 1988, Brown et al. 1989, Buroker et al. 1990). The whole sequence of the cytochrome b gene for A. transmontanus, as well as partial sequences of the DNA extraction, amplification, and sequencing same gene for Scaphirhynchus platorynchus and Polyodon spathula were published recentely DNA was isolated from each sample using a stan- (Brown et al. 1989, Normark et al. 1991). Partial se- dard phenol preparation (Hillis et al. 1990, DeSalle quences of the same gene for A. brevirostrum, A. et al. 1993). We examined partial sequences of three oxyrinchus, Scaphirhynchus albus, and S. suttkusi ribosomal genes (two mitochondrial and one nucle- were submitted by W. Schill into GenBank under ar) and a partial sequence of cytochrome b. PCR numbers Z22822, L35111, L35110, and L35112, re- products were prepared for DNA sequencing in spectively. Although acipenseriforms were includ- several ways. In all cases the nuclear 18S rDNA ed in a higher level phylogenetic analysis based on fragments were GeneCleaned (BIO 101; Palumbi et cytochrome b sequence (Normark et al. 1991), no al. 1991) and directly sequenced. PCR products of direct evidence on the utility of other gene regions the mitochondrial genes (12S, 16S, and cytochrome for phylogenetic analysis is available. b) were either GeneCleaned and directly se- Because phylogenetic divergence within the Aci- quenced or cloned into the TA vector (INVITRO- penseriformes is potentially broad, based on the GEN) and sequenced (in such cases, at least two fossil record (e.g., Grande & Bemis 1991, Bemis et clones for each were used to establish the se- al. 1997), no single gene region can be assumed to be quence). We used the following primers: in the 18S adequately broadly informative on all phylogenetic gene region, 18sai0.7 (5′-ATTAAAGTTGTTGC- levels as a source of characters. Consequently we GGTTT-3′) and 18sai0.79 (5′-TTAGAGTGCTY- chose to examine several gene regions as potential AAAGC-3′) (Wheeler et al. 1993), in the 12S gene sources of characters, including well characterized region, 12SA (5′-GGTGGCATTTTATTTTATT- gene regions from mitochondrial DNA (16S rDNA, AGAGG-3′) and 12SB (5′-CCGGTCTGAACTC- 12S rDNA, and cytochrome b) and one nuclear AGATCACGT-3′) (Kocher et al. 1989, Hedges et gene region (18S rDNA). Below we discuss each of al. 1993b), in the 16S gene region, 16SA (5′-CG- these four gene regions. CCTGTTTACCAAAACAT-3′) and 16SB (5′-CC- GGTCTGAACTCAGATCACGT-3′) (Palumbi et al. 1991), and in the cytochrome b region, H15149 Materials and methods (5′-AAACTGCAGCCCCTCAGAATGATATT- TGTCCTCA-3′) (Kocher et al. 1989) and L14724 Specimens (5′-CGAAGCTTGATATGAAAAACCATCG- TTG-3′) (Meyer et al. 1990). All sequencing was Species used in this study and location of the fishes performed using the Sequenase system (U.S. Bio- are listed in Table 5. With three exceptions (Acipen- chemicals) and double stranded templates. The se- ser brevirostrum, A. mikadoi, and A. oxyrinchus), quences reported in this paper have been deposited blood samples were taken, mixed with buffer (100 in the EMBL Nucleotide Sequence Database (ac- mM Tris, 100 mM EDTA, and 2% SDS; 0.5 ml of cession no. X95003–X95061). blood and 5 ml of buffer), and the blood cells lysed in this solution were kept in a freezer at −70°C. Most Russian specimens examined were the same

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110969 EBFI ART.NO 1621 (349) ORD.NO 231349.F 142

DNA sequence alignment and phylogenetic analysis

We used equal weights for all nucleotide positions in all analyses. When multiple parsimonious trees were obtained for a particular analysis, successive weighting based on the retention index was used to choose among these multiple parsimonious trees (Carpenter 1988). DNA sequences for the mito- chondrial 16S and 12S rRNA regions and the nucle- ar 18S rRNA regions were aligned using the pro- gram MALIGN (Wheeler & Gladstein 1993). Gap costs were varied in order to explore the effects of alignment parameters on phylogenetic inference (the results of varying alignment parameters on phylogenetic inference are discussed in detail in Fitch & Smith 1983, Gatesy et al. 1993, Hillis et al. 1994, Wheeler 1995, Wheeler et al. 1995). In most

Figure 2. A phylogenetic tree for a combined (18sai0.7 plus cases our alignments were extremely stable (i.e., 18sai0.79; 229 bp) region of fishes: eight acipenseriform species alignment columns did not change by altering gap studied, Polypterus senegalus, Amia calva; four chondrich- costs) and this stability suggests a low level of ‘align- thyans, Notorynchus cepedianus (Hexanchiformes, Hexanchi- ment ambiguity’ (Gatesy et al. 1993). Consequently, dae; Bernardi & Powers 1992), Echinorhinus cookei (Squali- the methods of ‘culling’ (Gatesy et al. 1993) and fomes, Squalidae; Bernardi & Powers 1992, Bernardi et al. 1992, M91179, GenBank), Squalus acanthoides (Squalifomes, Squal- ‘eliding’ (Wheeler et al. 1995) were not applied to idae; Bernardi & Powers 1992, Bernardi et al. 1992, M91181, Gen- infer alignment. It was trivial to align cytochrome b Bank), and Rhinobatos lentiginosus (Stock & Whitt 1992, sequences, which were also performed using MA- M97576, GenBank); Latimeria chalumnae (Stock et al. 1991b, LIGN with a gap cost of 8. Parsimony trees for each L11288, GenBank); and two teleosts, Fundulus heteroclitus (Cy- of the four individual gene regions were generated prinodontiformes, Fundulidae; Bernardi et al. 1992, M91180, GenBank), and altivelis (, Scor- separately using the PAUP 3.1 program (Swofford paenidae; M91182, GenBank). Squalus acanthias and Rhinoba- 1993) to examine the signal inherent in each gene tos lentiginosus were used as outgroups. region. Sequence alignments using a gap cost of 8 were arbitrarily chosen and were combined (Kluge Outgroup choice 1989, Ernisse & Kluge 1993) into a single data ma- trix. Phylogenetic hypotheses were generated from We chose Polypterus senegalus, a representative of this combined data matrix using PAUP. The degree a lineage often considered to be the sister group of of support for particular nodes in these trees was Acipenseriformes plus Neopterygii (Patterson examined using the Bremer support index (Bremer 1982), as our outgroup. Because the use of multiple 1988, Donoghue et al. 1993, Kallersjo et al. 1993). outgroups is recommended in phylogenetic analys- es (Watrous & Wheeler 1981), we also used Amia calva (Amiidae), generally regarded as the living Results and discussion sister species of teleosts (see Patterson 1973), as an outgroup. In the analysis of the partial sequence of Gene regions the 18S gene we used two chondrichthyan species, Squalus acanthias and Rhinobatos lentiginosus 18S rDNA (Bernardi et al. 1992, M91179; Stock & Whitt 1992, The 18S rRNA gene is relatively slowly evolving in M97576), as outgroups. vertebrates and has been useful at higher taxonom- ic levels (e.g., Stock et al. 1991a). We used two se-

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110969 EBFI ART.NO 1621 (349) ORD.NO 231349.F 143 quencing primers in our analyses, 18sai0.7 and there are three nucleotides, TCG, whereas in tele- 18sai0.79. These primers are in a region of the 18S osts there are 7 nucleotides, TTCTCCT or gene that varies in insects and other organisms TCTTTCT, and in species studied by us, only a part (Wheeler et al. 1993). The 18sai0.7 sequences were of the latter sequence, CCT. invariant in all acipenseriform species investigated, whereas the 18sai0.79 fragment was variable at sev- 16S mitochondrial rDNA eral positions. A low degree of 18S rRNA sequence We obtained sequences for two parts of this gene: divergence between Scaphirhynchus and Polyodon (1) a 147 nucleotide sequence using the 16SA primer was reported previously (Stock et al. 1991a). (146 nucleotides for Amia calva and Polypterus se- The phylogenetic tree for combined data sets for negalus), and (2) a 169 nucleotide sequence with the both fragments of the 18S gene for all fish species 16SB primer (164 nucleotides in Amia calva). Both investigated so far is presented in Figure 2. Species regions were highly conserved, although there were used and origin of the sequences are explained in differences in the 3′-part of the 16Sb fragment. the legend to this figure. Echinorhinus cookei and Rhinobatos lentiginosus were chosen as outgroups. 12S mitochondrial rDNA The tree statistics are shown in Table 6. Two overlapping short stretches of 12S mtrDNA, There is a high degree of similarity between the 12SA and 12SB, were sequenced. The final contig- gene fragments under consideration in all four uous sequence consisted of 183 nucleotides in Huso chondrichthyans and Latimeria chalumnae, and dauricus and Pseudoscaphirhynchus kaufmanni, they differ from these fragments in - 184 nucleotides in Polyodon spathula, A. mediros- gyans. There are two putative synapomorphies of tris, A. baerii, Scaphirhynchus albus, and Amia cal- acipenseriformes in the 18sai0.7 region: (1) all aci- va, and 185 nucleotides in Polypterus senegalus. The penserids and Polyodon spathula had an insertion 12S region is more variable than the 16S regions and of A between 684 and 685 comparatively to the ho- yields several phylogenetically informative charac- mologous sequence of L. chalumnae, and (2) a T ters for examining higher level relationships (see between positions 773 and 774 relative to L. cha- below). Extremely low levels of variability, how- lumnae. Also, a change of A to T in position 771 (L. ever, exist within the genera Acipenser and Huso. chalumnae) seems to be synapomorphic for all aci- penseriform species. The most variable region of Cytochrome b 18sai0.79 region in all groups of fishes examined so The amplified region consisted of 270 base pairs, far lies between T and G in position 851 and 855 (L. from the 7th to 97th codons according to Normark chalumnae). In chondrichthyans and L. chalumnae et al. (1991). Most variation occurs at the third posi-

Table 6. Tree statistics.

Gene Number of Apomorphies, Number of Number of Number of Consistency Retention characters total number phylo- trees steps index index genetically informative characters

(18sai0.7+ 18sai0.79) fragment 241 39 24 6 47 0.94 0.98 12S gene fragment 189 51 10 6 61 0.71 0.67 16S gene fragment 318 74 23 2 86 0.82 0.81 18S gene fragment 229 12 7 3 14 0.88 0.91 Cytochrome b gene fragment 270 163 56 3 187 0.62 0.54 Combined molecular characters 1006 300 96 1 354 0.65 0.59 Cytochrome b with additional species of Acipenser 270 163 56 2 216 0.54 0.52

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110969 EBFI ART.NO 1621 (349) ORD.NO 231349.F 144 tions of codons. For comparable lengths of the 16S and 12S sequences, the cytochrome b gene for the species examined by us has from three to four times more nucleotide changes.

Generic relationships within Acipenseriformes

Alignment and phylogenetic inference First, we examined phylogenetic signal in the indi- vidual gene character sets by constructing separate for each of the four genes studied. Sec- ond, due to the small number of apomorphic char- acters for each gene, we used a combined approach (Miyamoto 1985, Kluge 1989, Ernisse & Kluge 1993) to infer phylogenetic relationships. We used aggressive alignment parameters for the program MALIGN (build; treeswapping; random sequence addition; Wheeler & Gladstein 1993) in our computer searches. We avoided rearranging computer generated alignments based on eye judg- ment because of the arbitrary and inherently non- repeatable nature of this approach and because the Figure 3. Consensus trees for individual gene regions examined choice of gap:change cost ratios in DNA sequence for the 12S mtrDNA (189 bp), 16S mtrDNA (318 bp), 18S rDNA (229 bp), and cytochrome b genes (270 bp). Amia calva and Pol- alignment can greatly affect final alignment (Fitch ypterus senegalus are outgroups. The tree statistics are in Table 6. & Smith 1983, Waterman et al. 1992, Gatesy et al. 1993). We performed several alignments with var- ying gap:change ratios, and found alignments for all their positions in the sequences for each gene are three rRNA were very stable. There were few align- shown in Figure 4. ment ambiguities as judged by comparing align- In general, each gene tree alone showed low lev- ments generated at gap:change costs of 2, 4, 8 or 16. els of resolution (Figure 3). Successive weighting of The phylogenetic hypotheses generated by these the individual character sets did not result in the various alignments were congruent, further sup- choice of a single or fewer trees. Although charac- porting our notion that the alignments are very sta- ter congruence was high as indicated by the rela- ble. tively high consistency and retention indices, the number of informative characters for each charac- Individual gene trees ter set was so low (Figure 4) that resolution of only a We report three consensus gene trees from se- few nodes in each single gene tree was demonstrat- quences aligned using a gap:change ratio of 4 for the ed. three rRNA genes (Figure 3). As noted above, cyto- One general observation, however, is that Aci- chrome b sequences were aligned with a gap cost of penser was not found to be monophyletic in any of 8 and the resulting gene tree is also shown in Figure the four gene trees (Figure 3). For 12S mtDNA, Hu- 3. The tree statistics for each of the gene trees, in- so dauricus clustered with A. ruthenus, and no char- cluding the number of apomorphies and phyloge- acters were found that hypothesized the remaining netically important characters, is given in Table 6. species of Acipenser as a group. For 16S mtDNA, The phylogenetically informative characters and Polyodon spathula clustered with the four species of Acipenser surveyed, and no character was found

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Figure 4. Phylogenetically informative characters (nucleotide composition at variable sites) for four gene regions studied. Numbers above the sequences refer to base pair position from the first base in the amplified fragments. The first position for the amplified fragment for 12S (No. 35) corresponds to No. 570 in the published 12S sequence for Latimeria chalumnae (Hedges et al. 1992), in 16S (No. 30) corresponds to No. 176 for L. chalumnae (Hedges et al. 1992), in 18S (No. 33) corresponds to No. 682 for L. chalumnae (Stock et al. 1991b), and in cytochrome b gene (No. 6) corresponds to No. 25 for L. chalumnae (Normark et al. 1991). uniting Acipenser as monophyletic. In the 18S Combined molecular tree and comparison to exist- rDNA tree, there was again no evidence of mono- ing morphological hypothesis phyly of Acipenser. Finally, the cytochrome b tree The DNA sequence characters for the four gene re- grouped A. medirostris, A. transmontanus, A. ruthe- gions were combined into a data matrix and each nus and A. baerii with Huso dauricus. Acipenser me- character was given equal weight. One parsimony dirostris and A. transmontanus shared apomorphies tree was obtained using this combined molecular in the 18S rDNA and cytochrome b trees, and such a data matrix (Figure 5, Table 6). grouping was not ruled out by either of the other Findeis (1993, 1997) used osteological characters two trees. Pseudoscaphirhynchus and Scaphirhyn- for constructing a morphological phylogenetic hy- chus emerged as monophyletic in only one tree pothesis which focused on generic relationships (16S, Figure 3). among Acipenseriformes. Only a single parsimony tree resulted from his data set. He examined six spe- cies of acipenseriforms not included in our molecul- ar survey (Scaphirhynchus platorynchus, A. brevi-

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formation results from the small number of mole- cular characters that are pertinent to the Huso-Aci- penser sister group hypothesis. It is interesting, however, that many 19th century systematic studies placed Huso within Acipenser and that Huso was not elevated to a separate generic status until Brandt (1869). Later the generic status of Huso was still debated (for instance, Nikolukin 1970, Artyuk- hin 1995). Evidently, Huso warrants new attention from systematists. The sister relationship of Scaphirhynchus and Pseudoscaphirhynchus is strongly supported by the morphological data (Findeis 1993, 1997), but it is not supported by our molecular characters. In Figure 5 Scaphirhynchus emerged as the sister taxon of all other sturgeons, and Pseudoscaphirhynchus emerged as the sister taxon of Huso and Acipenser. This is an interesting difference between the two phylogenies because conventional pre-cladistic ideas about relationships within Acipenseridae sug- gest that Scaphirhynchus plus Pseudoscaphirhyn- Figure 5. The single parsimony tree obtained for the combined chus are basal members of the family (e.g., Zograf set of molecular characters. Amia calva and Polypterus senegalus 1887). The fact that Pseudoscaphirhynchus and Sca- are outgroups. Decay indices (Bremer 1988, Donoghue et al. 1993) are shown at each node. Numbers above the branches rep- phirhynchus did not group together is supported in resent bootstrap values computed by 1000 replications. The tree our combined tree by relatively high decay indices statistics are given in Table 6. (4 and 3 at the pertinent nodes in Figure 5) and bootstraps. We suspect that the traditional idea of rostrum, A. fulvescens, A. oxyrinchus, and Huso hu- this monophyly may be incorrect. so). He did not examine the osteology of three spe- cies studied by us (A. baerii, H. dauricus, and S. albus). Thus, there is incomplete overlap of taxa Relationships within the genus Acipenser surveyed by the two phylogenetic approaches, morphological and molecular. A further complica- For this investigation we used partial sequences of tion is that Findeis (1993, 1997) does not provide the cytochrome b genes of eight Eurasian and four evidence that Acipenser is monophyletic, whereas American species of the genus Acipenser. This data our total molecular data set does (Figure 5). set includes 7 species absent in the previous mole- Comparison of the osteological and molecular cular analysis because we did not sequence the ribo- trees shows two major differences: (1) the place- somal genes for these taxa. Taxa chosen represent ment of Huso dauricus and (2) the sister relation- all four species groups proposed by Artyukhin ship of Scaphirhynchus and Pseudoscaphirhynchus. (1995, see above). The result of phylogenetic analy- Findeis (1993, 1997) found that Huso is basal to the sis is presented in Figure 6, and tree statistics, in Ta- other Acipenseridae, and that the clade including ble 6. all other sturgeons was well supported by osteolog- Two parsimony trees were obtained. In the cyto- ical characters. Our combined molecular data, how- chrome b analysis, Acipenser is not monophyletic, ever, suggest that Huso dauricus is a sister-species and two main clades of species are seen in both to the genus Acipenser (Figure 5). Perhaps, this con- trees. Two western American species, A. mediros- flict between the molecular and morphological in- tris and A. transmontanus, group together and are a

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Figure 6. The two parsimony trees based on partial sequences (270 bp) of the cytochrome b gene regions from 11 species of Acipenser (representatives of all groups of species in Table 5), Huso dauricus, Pseudoscaphirhynchus kaufmanni, Scaphirhynchus albus, and Polyo- don spathula. As in the previous analyses, Amia calva and Polypterus senegalus were used as outgroups. The ploidy of fishes and the group number for species of Acipenser are given to the right of species names. The tree statistics is shown in Table 6. sister-group to A. mikadoi, whereas A. ruthenus is of two clades. The first clade includes the European basal to all these species. Except for the freshwater A. nudiventris grouped with the eastern American A. ruthenus, all other species of the clade are typ- A. oxyrinchus (and Huso dauricus, see discussion ically anadromous sturgeons. The difference in the above). Their sister-group consists of the European cytochrome b gene sequences between A. mikadoi A. gueldenstaedtii, Siberian A. baerii, and an east- and A. medirostris supports the previous assump- ern American species A. brevirostrum. These rela- tion based on DNA ploidy that A. mikadoi is a sep- tionships suggest that: (1) the Atlantic group of spe- arate species in spite of the fact that it is morpholog- cies is related to the Ponto-Caspian A. nudiventris; ically indistinguishable from A. medirostris (Bir- (2) a Ponto-Caspian European A. gueldenstaedtii is stein et al. 1993, Birstein 1994b). Unexpectedly, the closely related to a Ponto-Caspian Siberian A. bae- ancestral form of this group seems to be related to rii; (3) both Ponto-Caspian A. gueldenstaedtii and A. ruthenus. A. baerii are related to the eastern North American The second group is also unexpected. It consists

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A. brevirostrum; and (4) both clades show transat- 1991a, Hedges et al. 1993a). One surprising result of lantic relationships for the species. our study is the general lack of variability in the The position of a small clade consisting of A. stel- gene regions studied as compared to other latus and A. naccarii, is unresolved: in the first tree it groups, including teleosts (reviews in Meyer 1993, is grouped with the first main clade, whereas in the Meyer et al. 1993, Patarnello et al. 1994), some am- second tree it clusters with the second main clade phibians, most mammals and insects (for instance, (Figure 6). Traditionally, the octoploid A. naccarii Irwin et al. 1991, Hedges et al. 1993b, Wheeler et al. was considered to be closely related to the octo- 1993). The slow rate of molecular evolution in aci- ploid A. gueldenstaedtii (e. g., Tortonese 1989, Rossi penseriforms may be correlated with slow karyo- et al. 1991, Artyukhin 1995), but not to the tetraploid typic evolution in these fishes (see above). A. stellatus. The other group of fishes with a low rate of evolu- According to the tree in Figure 6, ploidization oc- tion in 18S and mitochondrial genes is Chondrich- curred at least three times within the Acipenser: two thyes (Bernardi & Powers 1992, Martin et al. 1992, octoploid ancestral forms were formed independ- Martin & Palumbi 1993). Slow evolution of the 18S ently in A. mikadoi-A. medirostris-A. transmonta- genes, as in acipenseriforms (see Figure 2), could be nus, A. gueldenstaedtii-A. baerii-A. brevirostrum, related to polyploidy (cryptoploidy) in these fishes and in A. stellatus-A. naccarii; two polyploidization (reviews in Schwartz & Maddock 1986, Birstein events followed resulting in the appearance of A. 1987, Stingo & Rocco 1991). But the nucleotide sub- mikadoi and A. brevirostrum. These data support stitution rate in the cytochrome b gene in sharks is our assumption (see above) that ploidization one sixth that of primates (Martin et al. 1992, Martin played a significant role in speciation within the & Palumbi 1993), and this characteristic cannot be Acipenser, but contradict a simple scheme of hypo- attributed to ploidy differences. Perhaps, it is thetical relationships of the species of Acipenser caused by differences in the rate of accumulation of published by Artyukhin (1995, see also Bemis et al. silent transversions (Martin & Palumbi 1993), but 1997). Except the close relatedness of A. transmon- this remains a peculiar problem. It is interesting tanus and A. medirostris, which is supported by that the sequence of the region of the 18S gene un- other molecular data (Brown et al. 1996), the other der discussion in sharks is more similar to that in the relationships in Artyukhin’s tree (Artyukhin 1995) coelacanth than to that in acipenseriforms (Figure are not supported by our molecular data. 2). We caution that the trees in Figure 6 are prelimi- It is evident that the molecular data (at least nary ones. Evidently, additional data should be ob- those presented here), as well as the cytogenetic da- tained for better resolution of relationships among ta (see above), have restrictions in application to the species. We have already sequenced longer re- the phylogeny of Acipenseriformes at the generic gions of the cytochrome b gene, as well as other level. Low levels of variability of the genes com- genes (Birstein & DeSalle 1997). Our data do show, monly used as phylogenetic tools (12S, 16S, 18S, and however, that phylogenetic relationships within the cytochrome b) suggests that some other, rapidly Acipenser can be reconstructed using even a partial evolving gene regions such as the mitochondrial sequence of the cytochrome b gene. control region (D-loop, Shedlock et al. 1992) or larger portions of the cytochrome b or other mito- chondrial structural genes (Normark et al. 1991) General lack of molecular variability among the gen- might be helpful for examining relationships among era of Acipenseriformes the genera of Acipenseridae. In the meantime, our results suggest that the cy- We initially chose the gene regions described above tochrome b gene could be used for investigation of because of the high degree of variability shown in relationships among species of Acipenser. The cyto- other taxa of comparable divergence times (Meyer chrome b data suggest that Acipenser is not mono- & Wilson 1990, Normark et al. 1991, Stock et al. phyletic (due to the insertion of Huso dauricus into

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110969 EBFI ART.NO 1621 (349) ORD.NO 231349.F 149 this group), as considered before. We hope that fu- 1993, 1997): Huso dauricus was a sister-species to ture analyses involving longer regions of the cyto- the genus Acipenser instead of being basal to all aci- chrome b gene and, possibly, some other protein penseriforms, and Scaphirhynchus and Pseudosca- coding genes will help to establish mono- or pol- phirhynchus did not form a monophyletic group. yphyly of this genus. (6) A partial sequence of the cytochrome b gene (270 bp) was used to examine relationships within the genus Acipenser. Seven additional species of Conclusions Acipenser were included in this part of the study (A. brevirostrum, A. gueldenstaedtii, A. mikadoi, A. (1) Little cytogenetic change has occured during the naccarii, A. nudiventris, A. oxyrinchus, and A. stel- evolution of Acipenseriformes. Polyodontidae and latus). The data support the hypothesis that octo- Acipenseridae presumably originated from a tetra- ploid species appeared at least three times within ploid ancestor whose karyotype consisted of 120 the Acipenser. Also they show close relationships macro- and microchromosomes with a DNA con- between the Eurasian A. ruthenus and the Pacific A. tent about 3.2–3.8 pg per nucleus. Tetraploidization mikadoi-A. medirostris-A. transmontanus, between of the 60-chromosome ancestor possibly occurred the European A. gueldenstaedtii, Siberian A. baerii, at the early times of evolution of the Acipenseri- and American A. brevirostrum, between two Eu- formes, probably, during the origin of this group in ropean species, A. stellatus and A. naccarii, as well the Mesozoic. as a possible trans-Atlantic relationship between (2) No conclusions regarding interrelationships the Eurasian A. nudiventris and American A. oxy- within Acipenseridae among Huso, Acipenser, Sca- rinchus suggesting limited utility of geographic lo- phirhynchus, and Pseudoscaphirhynchus can be cality as an indicator of relationship. made based on cytogenetic data. Divergence of these lineages of sturgeons occurred without pol- yploidization. Acknowledgements (3) Diversification within Acipenser was accom- panied by appearence of octoploids (according to We are very grateful to all colleagues who helped us karyotypic and DNA content data) and 16n-ploids to collect blood and tissue samples: Serge Gamalei (according to DNA content data). The octoploid (Moscow Aquarium, Moscow), Boris Goncharov 240-chromosome sturgeon species have about 240 (Institute of Developmental Biology, Russian chromosomes and may have originated independ- Academy of Sciences, Moscow), Evgenii Artyuk- ently in different geographic areas. The two 16n- hin (Central Laboratory of Regeneration of Fish ploid species, A. mikadoi and A. brevirostrum, may Resources, St. Petersburg), Francesco Fontana be the youngest species within the genus. (University of Ferrara), Herb Bollig (US Fish and (4) A study of partial sequences of genes from mi- Wildlife Service, ), Boyd Kynard (Na- tochondrial DNA (16S rDNA, 315 bp; 12S rDNA, tional Biological Survey, Massachusetts), John 189 bp, and cytochrome b, 270 bp) and of one nucle- North (Department of Fish and Wildlife, Oregon), ar gene region (18S rDNA, 230 bp) demonstrated and John Waldman (The Hudson River Founda- very low levels of variability in the eight acipenseri- tion, New York). We are also indebted to Paul Vra- form species surveyed (Polyodon spathula, Huso na for his kind permission to use his collection of dauricus, four species of Acipenser, Scaphirhynchus tissue samples. We are extremely thankful to Willy albus, and Pseudoscaphirhynchus kaufmanni). This Bemis for his patient reading of the manuscript and low variability is unusual for these genes, which are his important notes which allowed us to improve commonly used as phylogenetic tools. the manuscript immensely. (5) The molecular tree based on combined data from all four genes had two major departures from the existing morphological hypothesis (Findeis,

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